Reverse transcriptase is a DNA(RNA-DNA hybrid)-synthesizing enzyme using RNA as a template, which was discovered in Rausher murine leukemia virus (R-MLV) by Baltimore, 1970, and in Raus sarcoma virus (RSV) by Temin and Mizutani. This enzyme has been demonstrated in virions of RNA-containing tumor viruses and functions to make DNA complements of the RNA genoms. The resulting DNA, as a provirus, is integrated into the host cell chromosome and induces virus multiplication and transformation.
With the latest development of recombinant DNA techniques, reverse transcriptase has been widely used for synthesizing the complementary DNA (cDNA) from RNA and the demand for this enzyme has increased remarkably.
Reverse transcriptase has been produced using the plasma of chicks inoculated with avian myeloblastosis virus (AMV). However, this method requires the selection and conservation of chicken lines highly sensitive to AMV, as well as the use of special facilities and techniques for sampling at an appropriate stage of the disease. Because this method is complex, costly and time consuming, there has developed a need to establish simplified procedures to obtain reverse transcriptase, such as a method of using culture cells.
Purification of reverse transcriptase has been attempted by using culture cells of avian retrovirus as well as of mammalian retrovirus such as that obtained from the mouse and the monkey. However, these purification procedures require a large amount of materials for obtaining a suitable amount of the viruses, as well as a great deal of work and time for the treatment of these materials. Culture cells of mammalian retrovirus have not yet been successfully employed to produce the enzyme having a high specific activity.
It is therefore an object of the present invention to provide a cell line which can be cultured, propagated and transferred to produce retroviruses from which reverse transcriptase may be obtained.
It is a further object of the invention to extract the reverse transcriptase from the retroviruses and to subsequently purify the reverse transcriptase.